Diagnostic value of IgG antibody and stool antigen tests for chronic Helicobacter pylori infections in Ibb Governorate, Yemen

The stool antigen test (SAT) and the serum Helicobacter pylori (H. pylori) IgG antibody assays exhibit significant utility in the clinical diagnosis of H. pylori infection and in distinguishing between acute and chronic infections. The main objective of the current study was to identify the diagnostic value of serum H. pylori IgG antibody and SAT in the detection of H. pylori infections among chronic H. pylori-infected patients residing in Ibb Governorate, Yemen. 200 patients with H. pylori infection, confirmed through positive results in the serum immunochromatographic antibody test, were selected for H. pylori infection confirmation using serum H. pylori IgG antibodies and SAT across diverse hospitals, gastroenterology, and Hepatology clinics in Ibb Governorate. After the selection of patients, blood and stool specimens were obtained from all participants and underwent analysis via the Statistical Package for the Social Sciences (SPSS). The prevalence of H. pylori infection demonstrated variability based on the confirmatory tests, with rates of 54% for SAT and 78.5% for serum H. pylori IgG antibody, contrasting with a 100% prevalence observed in the screening serum immunochromatographic antibody test. Clinically, the study categorized H. pylori infections into four stages, whereby a significant proportion of patients (40.5%) exhibited positivity for both serum H. pylori IgG antibody and SAT, indicative of active chronic infections. The majority of positive cases only manifested serum H. pylori IgG antibody presence (chronic infections) at 38%, whereas 13.5% exclusively tested positive for SAT, corresponding to acute infections. Moreover, 88% of patients did not have either serum H. pylori IgG antibody or SAT (absence of infections) during confirmatory tests. Noteworthy is the study's approach employing multiple tests for H. pylori infection detection, focusing predominantly on chronic infections-prevailing types caused by H. pylori. The results revealed a significant association between serum levels of H. pylori IgG antibody and SAT results with the presence of diverse gastrointestinal symptoms among patients, which increased with long H. pylori infection durations.

The stool antigen test (SAT) and the serum Helicobacter pylori (H.pylori) IgG antibody assays exhibit significant utility in the clinical diagnosis of H. pylori infection and in distinguishing between acute and chronic infections.The main objective of the current study was to identify the diagnostic value of serum H. pylori IgG antibody and SAT in the detection of H. pylori infections among chronic H. pyloriinfected patients residing in Ibb Governorate, Yemen.200 patients with H. pylori infection, confirmed through positive results in the serum immunochromatographic antibody test, were selected for H. pylori infection confirmation using serum H. pylori IgG antibodies and SAT across diverse hospitals, gastroenterology, and Hepatology clinics in Ibb Governorate.After the selection of patients, blood and stool specimens were obtained from all participants and underwent analysis via the Statistical Package for the Social Sciences (SPSS).The prevalence of H. pylori infection demonstrated variability based on the confirmatory tests, with rates of 54% for SAT and 78.5% for serum H. pylori IgG antibody, contrasting with a 100% prevalence observed in the screening serum immunochromatographic antibody test.Clinically, the study categorized H. pylori infections into four stages, whereby a significant proportion of patients (40.5%) exhibited positivity for both serum H. pylori IgG antibody and SAT, indicative of active chronic infections.The majority of positive cases only manifested serum H. pylori IgG antibody presence (chronic infections) at 38%, whereas 13.5% exclusively tested positive for SAT, corresponding to acute infections.Moreover, 88% of patients did not have either serum H. pylori IgG antibody or SAT (absence of infections) during confirmatory tests.Noteworthy is the study's approach employing multiple tests for H. pylori infection detection, focusing predominantly on chronic infections-prevailing types caused by H. pylori.The results revealed a significant association between serum levels of H. pylori IgG antibody and SAT results with the presence of diverse gastrointestinal symptoms among patients, which increased with long H. pylori infection durations.
Helicobacter pylori is a Gram-negative bacterium, measuring 2-4 µm in length and 0.5-1 µm in width usually spiral-shaped but the bacterium can appear as a rod, while coccoid shapes appear after prolonged in vitro culture or antibiotic treatment 1,2 .
The incidence of H. pylori infection is nearly 50% of the global population, it affected by low hygienic conditions and poor socioeconomic status.In addition, the incidence of H. pylori infections in UK and USA were 13.4% and 27.1%, respectively.While in developing countries, the estimated prevalence was 90% and approximately 4.4 billion individuals worldwide were positive for H. pylori infections (more than half the world population).4][5][6] .
Gastritis defines as any inflammation of the gastric mucosa.Indeed, worldwide H. pylori infection is the most common cause of chronic gastritis; it is also the most important risk factor for peptic ulcer and its complications as well as for gastric cancer (GC).H. pylori causes progressive damage to the gastric mucosa and plays as

Materials and methods
The current research employed a cross-sectional design involving the collection of stool and blood specimens from 200 patients afflicted with H. pylori infections who had tested positive for rapid serum antibody of H. pylori between October 2019 and November 2020.Diagnosis of patients based on the detection of serum H. pylori antibodies via rapid diagnostic testing.Validation of the results was carried out utilizing the serum H. pylori IgG antibody (NovaLisa Helicobacter pylori IgG ELISA (HEL G0220) with 96 wells for determinations, manufactured by NOVA TEC IMMUNODIAGNOSTICA GMBH in Germany, exhibiting a specificity of 93.0% and sensitivity of 98%) for chronic infections, and the serum SAT (developed by Intech corporation, showcasing a sensitivity of 98.4% and a specificity of 98.8%) for acute infections at various hospitals, hepatology, gastrointestinal clinics, and different medical laboratories in Ibb Governorate, Yemen.All patients with confirmed H. pylori infections, irrespective of age or gender, were considered for inclusion in this study.Exclusion criteria comprised malignancies such as ovarian, endometrial, cervical, lung, breast, liver, gastric, pancreatic, and colorectal cancers, individuals with other gastrointestinal infections, and those who provided incomplete responses in the questionnaire.
A structured and reliable questionnaire was designed and distributed among all participants included in the study.This questionnaire encompassed the following sections: the demographic profiles of patients, including age, gender, Khat chewing (Catha edulis) smoking, educational background, dietary patterns, weight, height, familial medical history including incidences of H. pylori infections and peptic ulcer disease (PUD), duration of H. pylori infection, gastrointestinal symptoms including (nausea, vomiting, heartburn, and abdominal pain, along with the severity of these symptoms and the treatment administered for H. pylori.Blood samples of 5-8 ml were collected from patients, with serum segments isolated and preserved in sterile Eppendorf tubes for immediate usage in both rapid H. pylori antibody testing and serum H. pylori IgG antibody detection via an enzyme immunoassay (ELISA) test.Furthermore, stool specimens were promptly collected from all subjects for the prompt identification of H. pylori antigens in stool.Additionally, the body mass index (BMI) was calculated by dividing the individual's weight by (height) 2 .

Statistical analysis
A Statistical Package of Social Sciences (SPSS version 21) analyzed the data.The results presented as percentages, means, standard deviations (SD).In addition, Chi-square X 2 test was used for categorical variables.Furthermore, comparison was made by independent sample t-test and one-way ANOVA.P values < 0.05 were considered as statistically significant.

Ethical approval
The protocol of this work is approved by the Ethics Committee of Biological Sciences Department, Faculty of Science, Sana'a University, Yemen.This Ethics Committee agreed with the Helsinki Declaration for the Protection of Human Subjects.Informed consent was obtained from all the study participants.

Association of socio-demographic characteristics of study subjects with confirmatory H. pylori tests
Initially, upon examining the demographic profiles of 200 patients diagnosed with H. pylori infections and their correlation with confirmatory diagnostic procedures, a noteworthy rise in the average age of the patients was observed.Out of all patients, a majority of 108 individuals (54%) were female, while 92 (46%) were male.Additionally, 148 patients (74%) reported the consumption of Khat (Catha edulis), 48 (24%) were identified as smokers, 127 (63%) had a familial history of H. pylori infections, and 60 (30%) had familial occurrences of PUD.Notably, the results of the SAT revealed a significant statistical association at (P < 0.05) with various factors including body mass index (BMI), intake of fruits and vegetables, recurrent infections, treatment protocols, repeated infections, and endoscopic assessments.Conversely, no significant correlations were detected with age, gender, residency, Khat consumption, smoking habits, familial predisposition to H. pylori infections and PUD.Moreover, a statistically significant correlation at (P < 0.05) was established between positivity to serum H. pylori IgG antibodies and parameters such as age, BMI, smoking history, familial incidence of H. pylori infections

The diagnostic value of serum H. pylori IgG antibody and stool antigen test
Significantly, the study's findings indicated that the diagnostic value of SAT and serum H. pylori IgG antibody for the diagnosis of H. pylori infections exhibited notably higher values in the SAT-positive groups (25.37 ± 10.47 NTU/ml) compared to those in the SAT-negative groups (21.193 ± 8.22), indicating a statistically significant difference (P < 0.05), as depicted in Fig. 2.

Association between serum H. pylori IgG and SAT tests of H. pylori patients with gastrointestinal symptoms
Remarkably, the study revealed a significant correlation between SAT outcomes and gastrointestinal symptoms (inclusive of nausea, vomiting, heartburn, and abdominal pain), manifested by an increase in SAT positivity among symptomatic patients, as well as a significantly higher serum level of H. pylori IgG among symptomatic individuals.Additionally, the study indicated a linkage between H. pylori infection and vomiting, as evidenced by higher serum H. pylori IgG levels among patients experiencing vomiting as well as positivity to SAT (P < 0.05), as shown in Table 3.

Association between serum H. pylori IgG and SAT tests with severity of gastrointestinal symptoms
Clinically, the study's results indicated a statistically significant association between SAT outcomes and the severity of gastrointestinal symptoms, with a heightened rate of SAT positivity observed with increased symptom     Lastly, it was observed that the serum H. pylori IgG levels were significantly elevated (P < 0.05) among patients with an infection duration exceeding 3 years.On the other hand, the association between the SAT status of study participants and the duration of H. pylori infection indicated no substantial variance in SAT test outcomes when compared against the duration of H. pylori infection (P > 0.05), as shown in Table 5.

Discussion
Helicobacter pylori infection remains a significant global public health concern, with an estimated 4.4 billion individuals worldwide testing positive for H. pylori.Chronic H. pylori infections are commonly acquired during childhood (4).
The study results revealed a significant association between SAT outcomes and BMI, consumption of vegetables and fruits, recurrent infections, treatments, as well as endoscopic procedures.Conversely, no significant correlations were found with age, gender, place of residence, consumption of Khat (Catha edulis), smoking habits, familial history of H. pylori infections, or PUD.Notably, there was a significant correlation between serum H. pylori IgG antibody levels and age, BMI, smoking habits, familial history of H. pylori infections and PUD, recurrent infections, and treatments.These results are consistent with previous studies 18,19 that indicated the elderly population over 59 years to be most affected by H. pylori infections.A study conducted in Yemen 20 revealed a peak SAT positivity of 60% in the age range of 40-80 years and 45.6% in the age group of 19-40 years.Similarly, a study from Saudi Arabia 21 reported a higher prevalence of H. pylori infections using SAT in individuals over 50 years of age.
Regarding gender, the present study did not find any significant association between gender and seropositivity to H. pylori detected by serum H. pylori IgG antibody and SAT (P > 0.05), contrasting findings from another study 18 that reported a statistically significant link between H. pylori seropositivity and gender.The current study aligns with study by 22,23 which also found no significant gender-based differences in H. pylori infection.Furthermore, there was no significant disparity in H. pylori SAT outcomes based on gender (P > 0.05).
Consistent with a study by 24 , the findings of this study demonstrated a higher H. pylori positivity rate in patients who chewed Khat (Catha edulis).However, another study 25 contradicted these results, suggesting no significant difference in H. pylori infection rates based on Khat chewing (Catha edulis).Divergent outcomes have been documented in various studies regarding the correlation between H. pylori infection and smoking habits.
In a case-control study conducted in Nairobi, a higher prevalence of H. pylori infection was observed among among Khat chewers (Catha edulis), indicating a potential link between Khat (Catha edulis) consumption and susceptibility to gastrointestinal disorders 26 .Another study also suggested a greater occurrence of gastrointestinal disorders among Khat (Catha edulis) chewers, implying that Khat (Catha edulis) consumption might predispose individuals to gastrointestinal issues beyond just H. pylori infection 27 .Additionally, a study indicated a significant association between Khat (Catha edulis) chewing and duodenal ulcers, potentially attributed to stress related to Khat (Catha edulis) consumption 28 Furthermore, a separate study found a higher prevalence of H. pylori infection among individuals who engaged in Khat (Catha edulis) chewing 29 .
Various previous studies have highlighted smoking as a risk factor for different diseases, including gastric cancer and H. pylori infection 25,30,31 .The current study's findings revealed a significantly elevated presence of H. pylori IgG antibodies in smokers compared to non-smokers, with a statistically significant distinction.These findings contrasted with those from 32 , which indicated no significant difference in H. pylori infection rates between smokers and non-smokers (P > 0.05).
Concerning residency, the results of the current study aligned with those of 33 , indicating no significant difference in H. pylori infection rates between patients residing in urban and rural areas (P > 0.05).
Significantly, the study's findings highlighted a statistically significant variance in serum levels of H. pylori IgG antibodies and SAT concerning BMI (P < 0.05).These findings were in line with findings of many previous studies [34][35][36][37][38] .
The mechanisms of association between H. pylori and obesity are varied, involving gastrointestinal hormones such as ghrelin and leptin, pivotal in metabolic regulation and energy balance.Ghrelin, produced in the stomach, stimulates food intake, while leptin functions in the opposite direction.Studies have reported lower serum levels of leptin and ghrelin in patients with H. pylori infections.Moreover, insulin resistance, a significant risk factor for several metabolic disorders including obesity, may be influenced by H. pylori infection.Obesity's interaction  39,40 .
In relation to a family history of PUD, the present study's results concurred with those from 41 , showing an association between H. pylori infection and a PUD history (P < 0.05).However, these findings contradicted those of 42 , which indicated no statistically significant difference in H. pylori infection positivity based on a family history of PUD.The potential association of H. pylori infection with a family history of the infection might be attributed to inter-familial transmission, emphasizing close and intimate contact among family members as a crucial mode of H. pylori spread 43 .
In this study, the consumption of vegetables and fruits showed a significant association with H. pylori infection.This could be attributed to the unhealthy conditions in Yemen, inadequate access to clean water sources, and insufficient awareness regarding the necessity of thoroughly washing fruits and vegetables.Previous studies have suggested that fruits and vegetables could be exposed to contamination through irrigation with polluted water.Moreover, the main sources for H. pylori include soil, water, animal waste, and human feces [44][45][46] .
Furthermore, the study findings indicated that the prevalence of H. pylori infections detected through rapid tests stood at 100%, while serum H. pylori IgG positivity was at 78.5%, and the rate of H. pylori infection positivity using SAT among the participants was 54%.These results were consistent with a study of 47 , which reported IgG seropositivity for H. pylori in 48.3% of the study subjects and a SAT positivity rate of 28.2%.A study conducted in Yemen 20 , showed a blood antibody test positivity of 72% and SAT positivity of 49%.Other studies 19,48 indicated higher serum antibody positivity compared to stool antigen positivity.However 49 found a SAT positivity rate of 82%, while 50 revealed a lower SAT positivity rate of 32.22% among participants.The detection of H. pylori stool antigen and UBT are reliable indicators of an active infection 51 .On the other hand, serological tests for H. pylori IgG antibodies denote a past or chronic infection, with a decrease in H. pylori shedding observed with the progression of chronic infections, a pattern often seen in elderly individuals 52 .
The current study revealed distinct stages of H. pylori infections: 16 (8%) of the patients were negative for both serum H. pylori IgG antibody and SAT (no infections), 27 (13.5%)were only SAT positive (acute infections), 76 (38%) were only serum H. pylori IgG positive for both the serum H. pylori IgG antibody and SAT (chronic infections), and 81 (40.5%) were positive for both (active chronic infections).A study conducted in Ethiopia reported similar findings 53 .
Importantly, the study demonstrated that SAT and serum H. pylori IgG antibody had higher diagnostic value in SAT-positive groups (25.37 ± 10.47 NTU/ml) compared to SAT-negative groups (21.193 ± 8.22) at (P < 0.05).This aligns with previous studies 54,55 , reporting a seroprevalence of 78.4% using H. pylori serum IgG ELISA.However, the findings contrasted with a study 56 from KSA showing a lower prevalence of 28.3% using ELISA for H. pylori IgG detection.
The results of this study also indicated a significant association between SAT and gastrointestinal symptoms like nausea, vomiting, heartburn, and abdominal pain.Patients with symptoms had higher SAT positivity and serum H. pylori IgG levels.The study also showed that vomiting was particularly linked to H. pylori infection, with higher IgG levels among patients experiencing vomiting and positive SAT results.Similar associations were found in previous studies linking epigastric pain, heartburn, and vomiting to H. pylori infection [57][58][59][60] .
Moreover, the findings of the current study demonstrated a significant correlation between SAT positivity and the severity of gastrointestinal symptoms, with a higher rate of SAT positivity observed in individuals with more severe symptoms.Additionally, there was a statistically significant increase in the mean level of serum H. pylori IgG antibody among participants experiencing very severe symptoms.Another study explored the link between H. pylori infection and symptom severity, noting that moderate to severe symptoms were associated with RUT positivity compared to individuals with mild symptoms 61 .
Finally, patients with an H. pylori infection duration exceeding 3 years exhibited significantly elevated levels of serum H. pylori IgG.On the other hand, no significant difference was observed in the association between the SAT status of participants and the duration of H. pylori infection when compared.The prevalence of active chronic H. pylori infections was notably high, primarily attributed to key risk factors such as advancing age, smoking, Khat consumption (Catha edulis), family history of H. pylori infection, presence and severity of gastrointestinal symptoms, and prolonged duration of H. pylori infection.

19 Figure 2 .
Figure 2. The diagnostic value of serum H. pylori IgG antibody and stool antigen test.

Table 1 .
Association of Socio-demographic characteristics of study participants with confirmatory H. pylori tests.The results represented by frequency and percent %, H. pylori: Helicobacter pylori, PUD peptic ulcer disease, BMI body mass index, P ≤ 0.05 considered as significant.

Table 2 .
Prevalence of H. pylori infection according to different tests.The results represented by frequency and percent %, SAT Stool antigen test.
. Moreover, the mean level of serum H. pylori IgG antibodies was significantly higher in the group exhibiting very severe symptoms (P < 0.05), as shown in Table4.

Table 3 .
Association between serum H. pylori IgG and SAT tests of H. pylori patients with gastrointestinal symptoms.The results represented by frequency and percent %, Independent t test, X 2 : Chi square test *P ≤ 0.05 considered as significant.

Table 5 .
Association between serum H. pylori IgG and SAT tests with H. pylori infection duration.The results represented by frequency and percent %, X 2 : Chi square test, independent t test, *P ≤ 0.05 considered as significant.H. pylori infection is being explored, with indications that the immune milieu in obese individuals may support H. pylori survival with